In the present study, we for the first time demonstrated that miR-132/212 cluster contributes to the proliferation and inhibits apoptosis of PCSCs by targeting Ihh/PTHrP signaling pathway. obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster AZD8931 (Sapitinib) might serve as a novel target for bone diseases. test. All data were shown as the means. A statistical difference of em P /em 0.05 was considered significant. Results Isolation, purification and identification of PCSCs PCSCs were successfully isolated from the neonate rabbits distal epiphyseal growth plate using the methods described above. The morphological images of PCSCs were shown either under light microscope (Physique 1A) and immunostaining (Physique 1B). Fibroblast growth factor receptor-3 (FGFR-3) was recognized as a marker for PCSCs. Therefore, we detected its expression in the cultured PCSCs. The immunofluorescence image suggested positive FGFR-3 expression in PCSCs. Open in a separate window Physique 1 Isolation and identification of PCSCsPCSCs were isolated from the neonate rabbits distal epiphyseal growth plate and the morphology of PCSCs were observed under light microscope (A) and immunostaining with FGFR-3 (B). miR-132/212 cluster promotes growth and DNA synthesis of PCSCs In order to investigate the role of miR-132/212 cluster in the cell viability of PCSCs, miR-132/212 mimic, inhibitor and unfavorable control (NC) were transfected into PCSCs and cultured for different time points. MTT analysis showed that miR-132/212 mimic transfection for 24 h slightly, but significantly, increased cell viability of PCSCs. By contrast, miR-132/212 inhibitor suppressed PCSCs growth (Physique 2A). miR-132/212 inhibitor NC had no obvious effects on PCSCs growth. At 48 and 72 h, overexpression of miR-132/212 cluster further enhanced cell growth of PCSCs. Conversely, inhibition of miR-132/212 cluster decreased PCSCs growth (Physique 2A). Open in a separate window Physique 2 miR-132/212 cluster promotes growth and DNA synthesis of PCSCsAfter transfection with miR-132/212 mimic, inhibitor and unfavorable control (NC), MTT assay (A) and BrdU assay (B) were performed to measure the cell viability and DNA synthesis of PCSCs at 24, 48 and 72 h; * em P /em 0.05, ** em P /em 0.01. Next, we explored the role of miR-132/212 cluster in DNA synthesis of PCSCs using BrdU assay. After transfected, we found that up-regulation of miR-132/212 cluster for 24 h promoted the DNA synthesis of PCSCs (Physique 2B). Meanwhile, overexpression of miR-132/212 cluster further enhanced DNA synthesis of AZD8931 (Sapitinib) PCSCs. However, transfection with miR-132/212 inhibitor suppressed DNA synthesis in PCSCs in a time-dependent manner (Physique 2B). miR-132/212 cluster suppresses apoptotic death in PCSCs It is well established that cell apoptosis is usually closely associated with proliferation ability. Thus, we further examined the Mouse monoclonal to CK17 effect of miR-132/212 cluster on PCSCs apoptosis. Cytometry analysis showed that overexpression AZD8931 (Sapitinib) of miR-132/212 cluster significantly suppressed the numbers of apoptosis in PCSCs compared with negative controls, while down-regulation of miR-132/212 cluster elevated the apoptotic cell number in PCSCs (Physique 3). Moreover, miR-132/212 inhibitor NC had no obvious effects on PCSCs apoptosis. Taken together, these data showed that miR-132/212 cluster promotes PCSCs growth through AZD8931 (Sapitinib) inhibition of apoptosis. Open in a separate window Physique 3 miR-132/212 cluster suppresses apoptotic death in PCSCsAfter transfection with miR-132/212 mimic, inhibitor and unfavorable control (NC), flow cytometric analysis was performed to measure the cell apoptosis of PCSCs; * em P /em 0.05, ** em P /em 0.01. PTCH1 is usually a direct target of miR-132/212 cluster Bioinformatics analysis using online tools, including miRanda, PicTar and TargetScan, was performed to identify potential targets of miR-132/212 cluster. As a result, the 3UTR of PTCH1 gene was found to contain the conserved binding sites for miR-132/212 cluster..